Anti-siglec-15 antibodies and uses thereof

ABSTRACT

This disclosure relates to anti-Siglec-15 antibodies and uses thereof, in particular in the treatment of leukaemia, such as acute myeloid leukaemia.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part during work supported by funding fromthe European Community's Seventh Framework Programme ([FP7/2007-2013]under grant agreement no RG55610.

BACKGROUND

1. Field of the Disclosure

This disclosure relates to anti-Siglec-15 immunoconjugates, antibodies,antigen-binding fragments and uses thereof, in particular in thetreatment of acute myeloid leukaemia.

2. Background of the Disclosure

Acute myeloid leukaemia (AML) is a fatal illness where normalhaematopoiesis is replaced with a malignant proliferation of immatureblast cells. The symptoms, such as infection, bleeding, and anaemia, arerelated to this bone marrow failure and if left untreated will lead tothe death of the patient. AML affects around 2,000 people a year in theUK, with an increasing incidence as the population ages. Curativetreatment is highly intensive, which is arduous for the patient,expensive, and requires prolonged hospital admissions, presenting asignificant cost to the national health system. In addition, the use ofallogeneic bone marrow transplantation, either up-front or for salvagetherapy, represents a further cost in those patients for whom it isappropriate. Despite this, the majority of patients with AML still diefrom the disease.

Many drugs have been developed to treat AML. In addition to conventionalchemotherapy agents, therapeutic antibodies have been developed toselectively target AML blasts. Gemtuzumab ozogamicin (GO), targeting asialic acid binding immunoglobulin-like Siglec protein, CD33, isapproved for patients over 60 years of age (Bross P F et al., Clin.Cancer Res. 7(6):1490-6 (2001)).

CD33 is an antigen found on many AML blasts and GO is a toxin-conjugatedantibody that binds CD33. Recently, a randomized trial (AML15) reporteda disease-free survival advantage for patients treated with GO, mainlyrelated to a decreased incidence of relapse (Burnett A K et al., J.Clin. Oncol. 29(4):369-77 (2011)). However, approximately 10% cases ofAML do not express CD33 and GO is also associated with relatively rare,but potentially life-threatening side effects which includemyelosuppression, perhaps because CD33 is expressed on healthyhematopoietic stem cells (Taussig D C et al., Blood 106(13): 4086-92(2005)). In the AML15 trial this side effect appeared to have beenmanifested by the fact that patients treated with GO required moreplatelet transfusions and IV antibiotics. In other studies, treatmentwith GO has also resulted in hepatotoxicity as a dose limiting sideeffect (Mulford D., Semin. Hematol. 45(2):104-9 (2008)).

Although the existence of cancer stem cells for all malignancies is amuch debated topic, it is widely accepted that they are present inleukaemia. The first evidence for the existence of cancer stem cells wasdemonstrated in acute myeloid leukaemia. Bonnet and Dick isolated theCD34+CD38− subpopulation of leukemic cells and showed that these cellscould repopulate tumors in NOD/SCID mice (Bhatia M. et al., Proc. Natl.Acad. Sci. 94(10):5320-5 (1997)). Recent work also suggested thatleukaemia stem cell potential is present in the CD38+ compartment(Taussig D C et al., Blood 112(3): 568-75 (2008)). With a growingknowledge of their biology and unique identity, work is beginning totarget leukemic stem cells (Chan W I and Huntly B J, Semin. Oncol.35(4):326-35 (2008)). In leukaemia therapy, active research is targetingthis leukaemic stem cell subpopulation, however no drug is available todate that targets these cells specifically.

Siglecs are a family of immunoglobulin superfamily receptors that bindsialic acids (Varki A and Angata T, Glycobiology 16(1):1R-27R (2006)).They are commonly expressed on immune cells, in particular those immunecells of the myeloid lineage. Most Siglecs are inhibitory, but novelactivating Siglecs have been discovered recently, namely Siglec-14, -15and 16 (Angata T et al., FASEB J. 20:1964-1973 (2006), Angata T et al.,Glycobiology 17:838-846 (2007), Cao H et al., Eur. J. Immunol.38:2303-2315 (2008)). Different Siglecs have varying affinities fordistinct linkages of sialic acids, which are nine carbon based sugarsfound at the periphery of most mammalian cell surfaces. Siglec-15 is anewly described Siglec that is well conserved between species withrecognizable orthologues of the human sequence in zebrafish. Initialcharacterisation of Siglec-15 revealed α-2,6 linked sialic acids as itsligand, binding of which was dependent on an essential arginine residuein Siglec-15's N-terminal V-set immunoglobulin domain (Angata T et al.,2007). Siglec-15 has also been shown to bind an antigen highly expressedin a variety of tumors, sialyl-Tn (Angata T et al., 2007). Siglec-15 isunusual in that it is equipped with both negative and positivesignalling motifs. In the transmembrane domain, Siglec-15 can associatewith positive signalling adaptor molecules, such as DAP10, DAP12 and Fcreceptor common γ chain but, at the same time, Siglec-15 encodes acytoplasmic ITIM-like motif known as immunoreceptor-tyrosine basedswitch motif (ITSM) that generally mediates inhibitory signals(Shlapatska L M et al., J. Immunol. 166(9):5480-7 (2001)). The onlyother example of an immune receptor encoding dual signalling motifs isKIR2DL4, which associates with ITAM encoding adaptor Fc receptor commonγ chain (Miah S M et al., J. Immunol., 180(5):2922-32 (2008));Kikuchi-Maki A et al., J. Immunol., 174(7):3859-63 (2005)) and alsocontains an inhibitory ITIM motif in its own cytoplasmic tail (Faure Mand Long E O, J. Immunol., 168(12):6208-14 (2002)).

Interest in Siglecs as targets for treatment of leukaemia is growing.Nguyen et al profiled the majority of the known CD33rSiglecs (a majorsubfamily of Siglecs) for their expression on AML cells. (Nguyen D H,Exp. Hematol., 34(6):728-35 (2006)) They identified expression profilesof Siglecs characteristic of each major subgroup of AML as well aspatient-specific CD33rSiglec finger prints. A customized approach toleukaemia treatment has been proposed where full profiling of apatient's Siglec expression is followed by targeting a combination ofSiglecs. The advantage of targeting Siglecs is that most of them exhibitrapid endocytosis. Antibody-induced endocytosis of Siglec-5 and Siglec-9on the surface of U937 cells have similar half-lives of approximately100 minutes. However, no Siglec-based therapies have been developed forthe treatment of AML.

BRIEF SUMMARY

The invention identifies an association between Siglec-15 expression andleukaemia. The invention also provides for Siglec-15 immunoconjugates,antibodies, and antigen-binding fragments thereof and methods of usingsuch molecules to treat leukaemia, such as AML. Importantly, theSiglec-15 immunoconjugates, antibodies, and antigen-binding fragmentsprovide for therapeutics that avoid the side-effects associated withCD33-targeted AML therapies.

The invention provides for an isolated antibody or antigen-bindingfragment thereof that specifically binds to Siglec-15 comprising: a VHsequence at least 90%, 95% or 100% identical to the amino acid sequenceof SEQ ID NO:1; a VL sequence at least 90%, 95% or 100% identical to theamino acid sequence of SEQ ID NO:2; a VH-CDR1, VH-CDR2 and/or VH CDR3sequence identical to or identical to except for one, two, or threesubstitutions in each CDR relative to the VH-CDR1, VH-CDR2 and VH-CDR3sequences corresponding to SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5,respectively; a VL-CDR1, VL-CDR2 and/or VL CDR3 sequence identical to oridentical to except for one, two, or three substitutions in each CDRrelative to the VL-CDR1, VL-CDR2 and VL-CDR3 sequences corresponding toSEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; or VH-CDR1,VH-CDR2, VH CDR3, VL-CDR1, VL-CDR2 and VL CDR3 sequences identical to oridentical to except for one, two, or three substitutions in each CDRrelative to the VH-CDR1, VH-CDR2, VH CDR3, VL-CDR1, VL-CDR2 and VL CDR3sequences corresponding to SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

In particular embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VH sequence at least 90%, 95% or 100%identical to the amino acid sequence of SEQ ID NO:1 and a VL sequence atleast 90%, 95% or 100% identical to the amino acid sequence of SEQ IDNO:2.

In certain embodiments, an immunoconjugate, isolated antibody orantigen-binding fragment of the invention induces endocytosis ofSiglec-15 at a rate where the half-life of endogenous surface Siglec-15on human myelogenous leukemia K562 cells is less than about 5 minutes,less than about 4 minutes, or less than about 3 minutes

In certain embodiments, an isolated antibody or antigen-binding fragmentof the invention specifically binds to or competes with the sameSiglec-15 epitope as an antibody or antigen-binding fragment asdescribed above. In further embodiments, the isolated antibody orantigen-binding fragment of the invention specifically binds to the sameSiglec-15 epitope as an antibody or antigen-binding fragment thereofcomprising the VH and/or VL regions, or one or more CDR regions, of A9E8or competitively inhibits Siglec-15 binding by an antibody orantigen-binding fragment thereof comprising the VH and/or VL regions, orone or more CDR regions, of A9E8.

In certain embodiments, an antibody or antigen-binding fragment of theinvention is humanized, chimeric, fully human, a Fab fragment, a Fab′fragment, a F(ab)2 fragment, or a single chain Fv (scFv) fragment.

Also provided is a polypeptide comprising the VH and/or VL sequences orone or more of the CDR sequences of an antibody or antigen-bindingfragment of the invention. In other embodiments, provided is an isolatedcell producing the antibody, antigen-binding fragment, or polypeptide ofthe invention and a method of making the antibody, antigen-bindingfragment, or polypeptide of the invention, comprising (a) culturing anisolated cell producing the antibody or antigen-binding fragment; and(b) isolating an antibody, antigen-binding fragment, or polypeptide fromthe cultured cell.

Also provided is an immunoconjugate having the formula (A)-(L)-(C) or(C)-(L)-(A), wherein: (A) is an antibody or antigen binding fragmentthereof, or polypeptide of the invention; (L) is a linker; and (C) is acytotoxic agent; and wherein the linker (L) links (A) to (C).

The invention further provides for a composition comprising animmunoconjugate, antibody, antigen-binding fragment, or polypeptide ofthe invention and a carrier.

Also provided is an isolated polynucleotide comprising a nucleic acidencoding a VH or VL as described above. In particular embodiments, thepolynucleotide of the invention encodes an antibody comprising thenucleotide sequence SEQ ID NO:3 or SEQ ID NO:4. In further embodiments,provided is a vector comprising a polynucleotide of the invention.

Also provided is a method of treating acute myeloid leukaemia in asubject in need thereof, comprising administering to the subject aneffective amount of an immunoconjugate, antibody, antigen-bindingfragment or composition of the invention. In additional embodiments, amethod of treating acute myeloid leukaemia reduces the number ofleukemic stem cells.

Also provided is a method of reducing the side effects associated withtreating acute myeloid leukaemia in a subject in need thereof comprisingadministering to the subject an effective amount of an immunoconjugate,antibody, antigen-binding fragment or composition of the invention. Incertain embodiments, the side effect(s) are myelosuppression and/orhepatotoxicity.

Also provided is a method of reducing the number of blasts in an AMLpatient comprising administering to the patient an effective amount ofan immunoconjugate, antibody, antigen-binding fragment or composition ofthe invention. In additional embodiments, the blasts are CD33+ or CD33−.

Also provided is a method of treating acute myeloid leukemia in asubject having either low expression of CD33 or are CD33−, comprisingadministering to the subject an effective amount of an immunoconjugate,antibody, antigen-binding fragment or composition of the invention. Incertain embodiments, the low expression is due to treatment with aCD33-targeted therapy.

Also provided is a method for making acute myeloid leukemia therapeuticscomprising generating antibodies that bind Siglec-15, where thetherapeutics preferentially target AML blasts over normal peripheralblood leukocytes. In particular embodiments, the antibodies according tothis method are recombinant.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Siglec-15 transcript expression. (A) Four human Siglec-15 splicevariants were identified by RT-PCR using specific primers.Electrophoresis of RT-PCR products from DAMI (megakaryocytic cell line),HEL (erythroleukaemic cell line) and K562 (erythroleukaemic cell line)are shown (left). Exon maps of the transcripts were generated aftersequencing (right). Labelling reflects the number of extracellularimmunoglobulin like domains contained in each splice variant, e.g. “2D”means 2 domains, etc. For the one domain “1D” splice variant, twoisoforms were identified. “1D-A” encodes an extra short peptide fromexon 2, which is lacking in “1D-B”. (B) Results of RT-PCR analysisidentifying a Siglec-15 transcript in 6 of 7 different cell linestested. Only YT (natural killer-like leukaemic cell line) showed noSiglec-15 transcript. PMA or LPS stimulations over 48 hours up-regulatedexpression of the transcripts. GAPDH RT-PCR is shown underneath eachpanel to control for equal loading (0.4 kb). 293T cells were used as anegative control.

FIG. 2: Siglec-15-specific monoclonal antibodies. (A) Phage displaytechnology was employed to select for clones expressing single chainfragment variable (ScFv) binders to purified Siglec-15 and also cellsurface transfected Siglec-15. The variable domains (VH and VL) from twoselected phage ScFv's were cloned into a mouse IgG1 antibody backboneand expressed as complete mouse IgG1 antibodies. The resultantmonoclonal antibodies showed stronger binding to the CHO-pDisplaySiglec-15 stable cell line (unfilled histograms) than the parental CHOcell line (filled histograms) by flow cytometry. HA staining was used asa positive control. pDisplay constructs encode only the extracellulardomains of Siglec-15 with N-terminal HA tag and a PDGFR transmembranedomain. (B) HEK-293 DAP10 stable cell lines were transiently transfectedwith full length FLAG-Siglec-15 for detection by confocal microscopyusing monoclonal antibodies A9E8 and A4C9 (red). The cell surfacemarker, CD44 (rat antibody, blue) and endosomal marker, EEA1 (rabbitantibody, green) were used to define cell boundaries. Both antibodiesstained similarly as compared to the positive control (mouse anti-FLAGmonoclonal antibody). A HEK-293 expressing DAP10 stable cell line wasused because the membrane adaptor molecule DAP10 facilitated surfaceexpression of Siglec-15 (FIG. 3). (C) Specificity of A9E8 and A4C9 toSiglec-15 by FMAT (similar to confocal microscopy) comparing CHO stablecell lines encoding different Siglecs. ScFv versions of A9E8 and A4C9bound specifically to the Siglec-15 CHO stable cell line. (D) Westernblotting using A4C9 antibody. Lanes on the gel contain lysates ofuntransfected (UT) 293T cells and 293T cells transfected (T) with fulllength FLAG-Siglec-15 (right). The major band detected at ˜43 kDamatches the size of fully glycosylated Siglec-15 and is sensitive toEndo-H digestion. Weak background bands were observed at ˜50 kDa.

FIG. 3: Intracellular expression of Siglec-15. Daudi and K562 cells weresubjected to FACS analysis for Siglec-15 expression using the A9E8monoclonal antibody. FACS plots show forward scatter (FSC) on x-axis andeither isotype or Siglec-15 (A9E8) staining on y-axis. Percentagesindicate the proportion of live cell population with positive antibodystaining levels above background (Rectangular gates). Unfixed stainingshows surface expression (left), while fixed and permeabilized staining(right) reveal additional intracellular expression. For intracellularstaining, cells were fixed first in paraformaldehyde (30 minutes, roomtemperature), washed three times in PBS and then permeabilized in 0.5%Triton-100 (10 minutes, room temperature) and washed for a further threetimes in PBS. Mouse IgG1 monoclonal isotype control antibody (Dako) wasused in parallel. For unfixed cells, live cell gating was performedusing SyTox Red (Invitrogen) staining.

FIG. 4: Adaptor association of Siglec-15. FACS analysis of FLAG-taggedfull length Siglec-15 and -16 expression on the surface of HEK-293 cellsstably expressing different membrane adaptor molecules was assessed,each expressing N-terminal HA-tags. HA and Flag tags were co-stained.The x-axis shows surface adaptor expression levels (HA-FITC staining)and the y-axis shows surface Siglec expression (Flag-cy3) The percentageof double positive cells (shown in upper right quadrant) reflectsadaptor dependent Siglec surface expression. Mock transfection wascarried out using an empty FLAG-CMV vector. Untransfected HEK-293 cellswere also used as a negative control. Adaptor and Siglec pairings whichdemonstrated increase in surface Siglec detection are boxed.

FIG. 5: Endogenous Siglec-15 surface expression on leukaemic cell lines.(A) FACS plots of surface Siglec-15 protein expression on leukaemic celllines. The x-axis shows forward scatter (FSC) and the y-axis matchedisotype control or Siglec-15 staining. A9E8 was used for Siglec-15staining and mouse IgG1 monoclonal control antibody (Dako) as isotypecontrol. Live cell gating was performed using SyTox Red (Invitrogen)staining. Percentages indicate the proportion of the live cellpopulation staining positive at levels above background. Leukaemic celllines of the myeloid lineage are shown in the first two rows. First row:monocytic (U937 and SKM-1) and promonocytic (NOMO-1) cell lines. Secondrow: erythroleukaemic (K562 and HEL) and megakaryocytic (DAMI) celllines. Third row: B-cell lymphoma derived (Daudi and Raji) and T-cellacute lymphoblastic leukaemia (Jurkat) cell lines. (B) Western blotanalysis of Siglec-15 cytoplasmic tail specific rabbit antisera (left)and mouse anti-FLAG monoclonal antibody staining of untransfected 293Tcell lysates and lysates from 293T cells transfected with full lengthFLAG-Siglec-15 construct. A fully glycosylated Siglec-15 band was foundat 43 kDa. (C) Western blot analysis of lysates from four leukaemic celllines (K562, Raji, Jurkat and HEL) using both A4C9 monoclonal antibodythat recognizes the extracellular domains of Siglec-15 and Tail 1Aantisera that is specific for the cytoplasmic tail portion. Arrowsindicate bands from both blots that match the ˜43 kDa expected size forfully glycosylated Siglec-15.

FIG. 6: Siglec-15 is expressed only on a small number of a subset ofhealthy peripheral blood leukocytes. (A) Peripheral blood leukocytesobtained from healthy donors were used to analyse cell surface Siglec-15expression on different leukocyte cell types. Murine IgG1 monoclonalantibody (Dako) was used as isotype control and different cell markerswere used to differentiate between cell lineages: CD3 (T cells), CD19 (Bcells), CD56(NK cells) and CD14 (monocytes). Live cells were gated byremoving cells of low forward and side scatter values. Percentagesindicate proportions of live cells in each quadrant. The X-axiscorresponds to FITC labelled lineage marker staining and the y-axiscorresponds to matched isotype control of Siglec-15 staining. Nolineages stained for surface Siglec-15 except <4% of CD14+ monocytes(CD14). (B) Macrophages and dendritic cells were differentiated fromperipheral blood monocytes and used for cell surface Siglec-15detection. Markers (CD14 for macrophages and CD86) were used to ensuresuccessful differentiation. Histograms show unstained (clear) and markerstained (red) macrophages and dendritic cells. FACS plots show forwardscatter on the x-axis and either isotype or Siglec-15 (A9E8) staining onthe y-axis. Percentages indicate the percentage of the population withpositive staining above background levels. Macrophages and dendriticcells treated with or without LPS for 24 hours are labelled.

FIG. 7: High Siglec-15 surface expression on blasts from AML patients.AML patient blasts were used for studying surface Siglec-15 expression.Eight out of 10 patients showed significant Siglec-15 surface expressioncompared to healthy PBLs. Four examples are shown: (A) FACS analysis ofcells from an individual AML patient (Patient A) showing high levels ofSiglec-15 expression (35.7% of population), co-expressing with CD14 andHLA-DR. Mouse IgG1 isotype control was added for comparison. (B) FACSanalysis of cells from a second AML patient (Patient B), showing thatcells were negative for CD33 while exhibiting high Siglec-15 expressionin 20.2% of the cell populations. Histograms compare CD33 expression(unstained—red, stained—clear) and Siglec-15 expression (unstained—red,stained—clear) on the same sample. (C) FACS analysis of cells from athird patient (Patient C) exhibited 10% Siglec-15 expressing blasts,most of which were CD33+CD14+HLA-DR−. (D) FACS analysis of cells from afourth patient (Patient D) had 14% Siglec-15 positive blasts. Histogramsshow difference in Siglec-15 surface staining for larger (higher forwardscatter) and smaller (lower forward scatter) blasts (Isotype—red;Siglec-15—clear). (E) Summary of percentage of total circulating cellsexpressing Siglec-15 from either AML patients (dots) or Healthy donors(crosses). Horizontal lines indicate mean values.

FIG. 8: Endocytosis of surface Siglec-15. The rate of endocytosis forendogenously expressed surface Siglec-15 on K562 cells was measured byfollowing loss of surface A9E8 binding upon incubation at 37° C. A9E8staining was on ice for 30 minutes. Cells were then resuspended inserum-free media pre-warmed to 37° C. and incubated for varying lengthsof time at 37° C. Time points varied from 30 seconds to 15 minutes.Isotype control antibody (mIgG1 control, Dako) was used to measurebackground staining/fluorescence. Mean fluorescence above isotypestaining was calculated for different time points and expressed as apercentage of the maximum surface staining without incubation at 37° C.Plot (top) shows the fall of surface staining of Siglec-15 over 15minutes; x-axis is time measured in seconds and y-axis is percentage ofinitial fluorescence. An exponential decay curve fits the data pointswell, corresponding to a half-life of 174 seconds. A parallel experimentwas carried out using CHO cells stably expressing surface Siglec-15 in apDisplay construct that lacks the natural Siglec-15 transmembranedomains and cytoplasmic tail and instead encodes a PDGFR transmembranedomain. Steady levels of surface staining (shown by line of best fit)for the CHO-Siglec-15 stable cell line demonstrates that A9E8 was notdissociating from the cell surface significantly during the first 15minutes of incubation at 37° C.

DETAILED DESCRIPTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody which specifically binds toSiglec-15,” is understood to represent one or more antibodies whichspecifically bind to Siglec-15. As such, the terms “a” (or “an”), “oneor more,” and “at least one” can be used interchangeably herein.

“Siglec-15” is a type-I transmembrane protein comprising twoimmunoglobulin-like domains, a transmembrane domain containing a lysineresidue, and a short cytoplasmic tail. The extracellular domain ofSiglec-15 preferentially recognizes Neu5Acα2-6GalNAcα structures and hasimmune system activating activity.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide as disclosed herein can be of a size of about 3 or more, 5or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated as disclosed herein, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Other polypeptides disclosed herein are fragments, derivatives, analogs,or variants of the foregoing polypeptides, and any combination thereof.The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to an antibody which specifically binds to Siglec-15 asdisclosed herein include any polypeptides which retain at least some ofthe antigen-binding properties of the corresponding native antibody orpolypeptide. Fragments of polypeptides include, for example, proteolyticfragments, as well as deletion fragments, in addition to specificantibody fragments discussed elsewhere herein. Variants of an antibodywhich specifically binds to Siglec-15 as disclosed herein, includefragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Variants can occur naturally or be non-naturally occurring.Non-naturally occurring variants can be produced using art-knownmutagenesis techniques. Variant polypeptides can comprise conservativeor non-conservative amino acid substitutions, deletions or additions.Derivatives of a an antibody or antigen-binding fragment whichspecifically binds to Siglec-15 as disclosed herein are polypeptideswhich have been altered so as to exhibit additional features not foundon the native polypeptide. Examples include fusion proteins. Variantpolypeptides can also be referred to herein as “polypeptide analogs.” Asused herein a “derivative” of an antibody or antigen-binding fragmentwhich specifically binds to Siglec-15 refers to a subject polypeptidehaving one or more residues chemically derivatized by reaction of afunctional side group. Also included as “derivatives” are those peptideswhich contain one or more naturally occurring amino acid derivatives ofthe twenty standard amino acids. For example, 4-hydroxyproline can besubstituted for proline; 5-hydroxylysine can be substituted for lysine;3-methylhistidine can be substituted for histidine; homoserine can besubstituted for serine; and ornithine can be substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide can comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody or antigen-binding fragment which specifically binds toSiglec-15 contained in a vector is considered isolated as disclosedherein. Further examples of an isolated polynucleotide includerecombinant polynucleotides maintained in heterologous host cells orpurified (partially or substantially) polynucleotides in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofpolynucleotides. Isolated polynucleotides or nucleic acids furtherinclude such molecules produced synthetically. In addition,polynucleotide or a nucleic acid can be or can include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g., on a single vector, or in separate polynucleotideconstructs, e.g., on separate (different) vectors. Furthermore, anyvector can contain a single coding region, or can comprise two or morecoding regions, e.g., a single vector can separately encode animmunoglobulin heavy chain variable region and an immunoglobulin lightchain variable region. In addition, a vector, polynucleotide, or nucleicacid can encode heterologous coding regions, either fused or unfused toa nucleic acid encoding an antibody, or antigen-binding fragment,variant, or derivative thereof which specifically binds to Siglec-15.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally can include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter can be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide can be RNA, for example, in theform of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions can be associated withadditional coding regions which encode secretory or signal peptides,which direct the secretion of a polypeptide encoded by a polynucleotideas disclosed herein, e.g., a polynucleotide encoding a antibody orantigen-binding fragment, or variant or derivative thereof whichspecifically binds to Siglec-15. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the complete or “full length” polypeptide to produce a secreted or“mature” form of the polypeptide. In certain embodiments, the nativesignal peptide, e.g., an immunoglobulin heavy chain or light chainsignal peptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, can be used. Forexample, the wild-type leader sequence can be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

Disclosed herein are certain antibodies, or antigen-binding fragments,variants, analogs or derivatives thereof including engineered antibodymolecules or fragments that bind antigen in a manner similar to theantibody molecules as described. Also disclosed are immunoconjugatescomprising such antibodies, or antigen-binding fragments, variants,analogs or derivatives thereof.

The terms “antibody” and “immunoglobulin” can be used interchangeablyherein. An antibody (or a fragment, variant, or derivative thereof asdisclosed herein comprises at least the variable domain of a heavy chainand at least the variable domains of a heavy chain and a light chain.Basic immunoglobulin structures in vertebrate systems are relativelywell understood. See, e.g., Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of this disclosure.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody orantigen-binding fragment to selectively recognize and specifically bindepitopes on antigens. That is, the VL domain and VH domain, or subset ofthe complementarity determining regions (CDRs), of an antibody orantigen-binding fragment combine to form the variable region thatdefines a three dimensional antigen binding site. This quaternaryantibody or antigen-binding fragment structure forms the antigen bindingsite present at the end of each arm of the Y. More specifically, theantigen binding site is defined by three CDRs on each of the VH and VLchains.

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table I as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,or variant or derivative thereof which specifically binds to Siglec-15as disclosed herein are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivativesthereof include, but are not limited to, polyclonal, monoclonal, human,humanized, or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library. ScFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulinor antibody molecules encompassed by this disclosure can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

By “specifically binds,” it is generally meant that an antibody orfragment, variant, or derivative thereof binds to an epitope via itsantigen binding domain, and that the binding entails somecomplementarity between the antigen binding domain and the epitope.According to this definition, an antibody or fragment, variant, orderivative thereof is said to “specifically bind” to an epitope when itbinds to that epitope, via its antigen binding domain more readily thanit would bind to a random, unrelated epitope. The term “specificity” isused herein to qualify the relative affinity by which a certain antibodyor fragment, variant, or derivative thereof binds to a certain epitope.For example, antibody “A” may be deemed to have a higher specificity fora given epitope than antibody “B,” or antibody “A” may be said to bindto epitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody cancross-react with the related epitope.

An antibody or fragment, variant, or derivative thereof, or animmunoconjugate comprising an antibody or fragment, variant, orderivative thereof is said to competitively inhibit binding of areference antibody or antigen binding fragment to a given epitope if itpreferentially binds to that epitope to the extent that it blocks, tosome degree, binding of the reference antibody or antigen bindingfragment to the epitope. Competitive inhibition can be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody or fragment, variant, or derivative thereof can be said tocompetitively inhibit binding of the reference antibody or antigenbinding fragment to a given epitope by at least 90%, at least 80%, atleast 70%, at least 60%, or at least 50%.

Antibodies or antigen-binding fragments, variants or derivatives thereofas disclosed herein can also be described or specified in terms of theircross-reactivity. As used herein, the term “cross-reactivity” refers tothe ability of an antibody or fragment, variant, or derivative thereof,specific for one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as the

Antibody fragments including single-chain antibodies can comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded are antibodies or antigen-binding fragments also comprising anycombination of variable region(s) with a hinge region, CH1, CH2, and CH3domains. Antibodies, or antigen-binding fragments thereof disclosedherein can be from any animal origin including birds and mammals. Theantibodies can be human, murine, donkey, rabbit, goat, guinea pig,camel, llama, horse, or chicken antibodies. In another embodiment, thevariable region can be condricthoid in origin (e.g., from sharks). Asused herein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulins and that do not express endogenousimmunoglobulins, as described for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. an antibody orfragment, variant, or derivative thereof, comprising a heavy chainportion comprises at least one of: a CH1 domain, a hinge (e.g., upper,middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain,or a variant or fragment thereof. For example, an antibody or fragment,variant, or derivative thereof can comprise a polypeptide chaincomprising a CH1 domain; a polypeptide chain comprising a CH1 domain, atleast a portion of a hinge domain, and a CH2 domain; a polypeptide chaincomprising a CH1 domain and a CH3 domain; a polypeptide chain comprisinga CH1 domain, at least a portion of a hinge domain, and a CH3 domain, ora polypeptide chain comprising a CH1 domain, at least a portion of ahinge domain, a CH2 domain, and a CH3 domain. In another embodiment, anantibody or fragment, variant, or derivative thereof comprises apolypeptide chain comprising a CH3 domain. Further, an antibody orfragment, variant, or derivative thereof for use in the disclosure canlack at least a portion of a CH2 domain (e.g., all or part of a CH2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) canbe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

The heavy chain portions of an antibody or fragment, variant, orderivative thereof as disclosed herein can be derived from differentimmunoglobulin molecules. For example, a heavy chain portion of apolypeptide can comprise a CH1 domain derived from an IgG1 molecule anda hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. The light chainportion comprises at least one of a VL or CL domain.

Antibodies or antigen-binding fragments, variants, or derivativesthereof disclosed herein can be described or specified in terms of theepitope(s) or portion(s) of an antigen that they recognize orspecifically bind. The portion of a target antigen which specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target antigen, e.g., apolysaccharide can comprise a single epitope, but typically comprises atleast two epitopes, and can include any number of epitopes, depending onthe size, conformation, and type of antigen.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit. The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which can beintact, partial or modified) is obtained from a second species. In someembodiments the target binding region or site will be from a non-humansource (e.g. mouse or primate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site. Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

The terms “fusion protein” or “chimeric protein” or descriptions of aprotein or polypeptide comprising two moieties that are “fused,” referto a first amino acid sequence linked to a second amino acid sequencewith which it is not naturally linked in nature. The amino acidsequences may normally exist in separate proteins that are broughttogether in the fusion polypeptide or they may normally exist in thesame protein but are placed in a new arrangement in the fusionpolypeptide. A fusion protein can be created, for example, by chemicalsynthesis, or by creating and translating a polynucleotide in which thepeptide regions are encoded in the desired relationship. As used herein,the terms “linked,” “fused” or “fusion” are used interchangeably.

The term “immunoconjugate” or “conjugate” as used herein refers to acompound or a derivative thereof that is fused or linked to a cellbinding agent (i.e., an anti-Siglec-15 antibody or fragment thereof) andis defined by a generic formula: C-L-A, wherein C=cytotoxin, L=linker,and A=cell binding agent or anti-Siglec-15 antibody or antibodyfragment. Immunoconjugates can also be defined by the generic formula inreverse order: A-L-C. An “immunoconjugate” comprises a targetingportion, or moiety, such as an antibody or fragment thereof whichretains antigen recognition capability, and an effector molecule, suchas a therapeutic moiety or a detectable label.

An “immunotoxin” is an immunoconjugate in which the therapeutic moietyis a cytotoxin. A “targeting moiety” is the portion of animmunoconjugate intended to target the immunoconjugate to a cell ofinterest, e.g., an anti-Siglec-15 antibody or antigen-binding fragmentthereof. Typically, the targeting moiety is an antibody, a scFv, a dsFv,an Fab, or an F(ab′)₂. The targeting moiety can also comprise, e.g., aFab′, a Fd, V-NAR domain, an IgNar, an intrabody, an IgGΔCH2, aminibody, a F(ab′)₃, a tetrabody, a triabody, a diabody, a single-domainantibody, DVD-Ig, Fcab, mAb², a (scFv)₂, or a scFv-Fc.

A “toxic moiety” is the portion of a immunotoxin which renders theimmunotoxin cytotoxic to cells of interest.

A “therapeutic moiety” is the portion of an immunoconjugate intended toact as a therapeutic agent and in some embodiments can be a “toxicmoiety.”

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to therapeuticmeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological change or disorder. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, a delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows,bears, and so on.

II. Antibodies or Antigen-Binding Fragments

Siglec-15 is a type-I transmembrane protein comprising twoimmunoglobulin-like domains, a transmembrane domain containing a lysineresidue, and a short cytoplasmic tail. The extracellular domain ofSiglec-15 preferentially recognizes Neu5Acα2-6GalNAcα structures and hasimmune system activating activity. Though previously identified ondendritic cells and macrophages, Siglec-15 is shown herein to beexpressed in high levels on the surface of AML blasts and in low levelson peripheral blood leukocytes.

One embodiment is directed to an antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15. In certain embodiments,the antibody or antigen-binding fragment thereof is A9E8. In furtherembodiments, the antibody or antigen-binding fragment thereof is animmunoconjugate comprising a Siglec-15 antibody or antigen-bindingfragment such as A9E8

The disclosure is further directed to an antibody or antigen-bindingfragment thereof which specifically binds to the same Siglec-15 epitopeas an antibody or antigen-binding fragment thereof comprising the heavychain variable region (VH) and light chain variable region (VL) regionof A9E8. Further included is an antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15 and competitively inhibitsSiglec-15 binding by an antibody or antigen-binding fragment thereofcomprising the A9E8 VH and/or VL; the A9E8 VH-CDR3 and/or A9E8 VL-CDR3;the A9E8 VH-CDR1, VH-CDR2, and VH-CDR3; the A9E8 VL-CDR1, VL-CDR2, andVL-CDR3; or the A9E8 VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, andVL-CDR3. An antibody is said to competitively inhibit binding of areference antibody to a given epitope if it preferentially binds to thatepitope to the extent that it blocks, to some degree, binding of thereference antibody to the epitope. Competitive inhibition may bedetermined by any method known in the art, for example, competitionELISA assays. An antibody may be said to competitively inhibit bindingof the reference antibody to a given epitope by at least 90%, at least80%, at least 70%, at least 60%, or at least 50%.

Due to the differential expression of Siglec-15 on AML blasts as opposedto normal peripheral blood leukocytes, Siglec-15 can be used to screenfor additional therapeutics, e.g., antibodies that bind Siglec-15 andimmunoconjugates comprising the anti-Siglec-15 antibodies, thatpreferentially target AML blasts and have minimal effects on normalperipheral blood leukocytes. In one embodiment, the Siglec-15therapeutic is an anti-Siglec-15 antibody or antigen-binding fragment.In another embodiment, the Siglec-15 therapeutic is an immunoconjugatecomprising an anti-Siglec-15 targeting moiety. In a further embodiment,the Siglec-15 therapeutic is a small molecule.

Methods of making antibodies are well known in the art and describedherein. Once antibodies to various fragments of, or to the full-lengthSiglec-15 without the signal sequence, have been produced, determiningwhich amino acids, or epitope, of Siglec-15 to which the antibody orantigen binding fragment binds can be determined by epitope mappingprotocols as described herein as well as methods known in the art (e.g.double antibody-sandwich ELISA as described in “Chapter 11—Immunology,”Current Protocols in Molecular Biology, Ed. Ausubel et al., v.2, JohnWiley & Sons, Inc. (1996)). Additional epitope mapping protocols can befound in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press(1996), which are both incorporated herein by reference in theirentireties. Epitope mapping can also be performed by commerciallyavailable means (i.e. ProtoPROBE, Inc. (Milwaukee, Wis.)).

In certain aspects, the disclosure is directed to an antibody orfragment, variant, derivative or immunoconjugate thereof whichspecifically binds to Siglec-15 and induces an enhanced rate ofSiglec-15 endocytosis. In certain embodiments, an antibody or fragment,variant, derivative or immunoconjugate thereof of the invention inducesendocytosis of Siglec-15 at a rate where the half-life of surfaceendogenous Siglec-15 on human myelogenous leukemia K562 cells is lessthan about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes,about 1 minutes or about 30 seconds.

Unless it is specifically noted, as used herein a “fragment thereof” inreference to an antibody refers to an antigen-binding fragment, i.e., aportion of the antibody which specifically binds to the antigen.

Accordingly, certain embodiments disclosed herein include ananti-Siglec-15 antibody, or antigen-binding fragment, variant, orderivative thereof, in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as reduced effectorfunctions, the ability to non-covalently dimerize, increased ability tolocalize at the site of a tumor, reduced serum half-life, or increasedserum half-life when compared with a whole, unaltered antibody ofapproximately the same immunogenicity. For example, certain antibodiesor antigen-binding fragment thereof described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH2 domain will be deleted.

Modified forms of anti-Siglec-15 antibodies or antigen-bindingfragments, variants, or derivatives thereof can be made from wholeprecursor or parent antibodies using techniques known in the art.Exemplary techniques are discussed elsewhere herein.

In certain embodiments both the variable and constant regions ofanti-Siglec-15 antibodies or antigen-binding fragments are fully human.Fully human antibodies can be made using techniques that are known inthe art and as described herein. For example, fully human antibodiesagainst a specific antigen can be prepared by administering the antigento a transgenic animal which has been modified to produce suchantibodies in response to antigenic challenge, but whose endogenous locihave been disabled. Exemplary techniques that can be used to make suchantibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592;6,420,140. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g.,phage display or other viral display systems, as described in moredetail elsewhere herein.

Anti-Siglec-15 antibodies or antigen-binding fragments thereof asdisclosed herein can be made or manufactured using techniques that areknown in the art. In certain embodiments, antibodies or fragmentsthereof are “recombinantly produced,” i.e., are produced usingrecombinant DNA technology. Exemplary techniques for making antibodymolecules or fragments thereof are discussed in more detail elsewhereherein.

In certain anti-Siglec-15 antibodies, the Fc portion can be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain can reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it can be that constant region modifications moderatecomplement binding and thus reduce the serum half-life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region can be used to modify disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. The resultingphysiological profile, bioavailability and other biochemical effects ofthe modifications, such as localization, biodistribution and serumhalf-life, can easily be measured and quantified using well knownimmunological techniques without undue experimentation.

In certain embodiments, anti-Siglec-15 antibodies or antigen-bindingfragments, variants, immunoconjugates or derivatives thereof will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In one embodiment, anti-Siglec-15 antibodies orantigen-binding fragments, variants, immunoconjugates or derivativesthereof are modified to reduce their immunogenicity using art-recognizedtechniques. For example, antibodies can be humanized, de-immunized, orchimeric antibodies can be made. These types of antibodies are derivedfrom a non-human antibody, typically a murine or primate antibody, thatretains or substantially retains the antigen-binding properties of theparent antibody, but which is less immunogenic in humans. This can beachieved by various methods, including (a) grafting the entire non-humanvariable domains onto human constant regions to generate chimericantibodies; (b) grafting at least a part of one or more of the non-humancomplementarity determining regions (CDRs) into a human framework andconstant regions with or without retention of critical frameworkresidues; or (c) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues. Such methods are disclosed in Morrison et al., Proc. Natl.Acad. Sci. 81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92(1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec.Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), andU.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all ofwhich are hereby incorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes (see, e.g., WO9852976A1,WO0034317A2). For example, VH and VL sequences from the startingantibody are analyzed and a human T cell epitope “map” from each Vregion showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative VH and VL sequences are designed comprising combinations ofamino acid substitutions and these sequences are subsequentlyincorporated into a range of binding polypeptides, e.g.,Siglec-15-specific antibodies or antigen-binding fragments thereofdisclosed herein, which are then tested for function. Complete heavy andlight chain genes comprising modified V and human C regions are thencloned into expression vectors and the subsequent plasmids introducedinto cell lines for the production of whole antibody. The antibodies arethen compared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Anti-Siglec-15 antibodies or antigen-binding fragments, variants,immunoconjugates or derivatives thereof can be generated by any suitablemethod known in the art. Monoclonal antibodies can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, 2nd ed. (1988)

DNA encoding antibodies or antibody fragments (e.g., antigen bindingsites) can also be derived from antibody libraries, such as phagedisplay libraries. In a particular, such phage can be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with scFv, Fab, Fv OE DAB(individual Fv region from light or heavy chains) or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Exemplary methods are set forth, forexample, in EP 368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R.and Chames, Immunol. Today 21:371 (2000); Nagy et al. Nat. Med. 8:801(2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui etal., J. Mol. Biol. 315:1063 (2002), each of which is incorporated hereinby reference. Several publications (e.g., Marks et al., Bio/Technology10:779-783 (1992)) have described the production of high affinity humanantibodies by chain shuffling, as well as combinatorial infection and invivo recombination as a strategy for constructing large phage libraries.In another embodiment, ribosomal display can be used to replacebacteriophage as the display platform (see, e.g., Hanes et al., Nat.Biotechnol. 18:1287 (2000); Wilson et al., Proc. Natl. Acad. Sci. USA98:3750 (2001); or Irving et al., J. Immunol. Methods 248:31 (2001)). Inyet another embodiment, cell surface libraries can be screened forantibodies (Boder et al., Proc. Natl. Acad. Sci. USA 97:10701 (2000);Daugherty et al., J. Immunol. Methods 243:211 (2000)). Such proceduresprovide alternatives to traditional hybridoma techniques for theisolation and subsequent cloning of monoclonal antibodies.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. For example, DNA sequences encoding VH and VL regions areamplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues) or synthetic cDNA libraries. In certainembodiments, the DNA encoding the VH and VL regions are joined togetherby an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli andthe E. coli is infected with helper phage. Phage used in these methodsare typically filamentous phage including fd and M13 and the VH or VLregions are usually recombinantly fused to either the phage gene III orgene VIII. Phage expressing an antigen binding domain that binds to anantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead.

Additional examples of phage display methods that can be used to makethe antibodies include those disclosed in Brinkman et al., J. Immunol.Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persicet al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references and in the examples below, afterphage selection, the antibody coding regions from the phage can beisolated and used to generate whole antibodies, including humanantibodies, or any other desired antigen binding fragment, and expressedin any desired host, including mammalian cells, insect cells, plantcells, yeast, and bacteria. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawaiet al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043(1988) (said references incorporated by reference in their entireties).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988).

Fully human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly orby homologous recombination into mouse embryonic stem cells. Inaddition, various companies can be engaged to provide human antibodiesproduced in transgenic mice directed against a selected antigen usingtechnology similar to that described above.

Fully human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903(1988). See also, U.S. Pat. No. 5,565,332.)

In another embodiment, DNA encoding desired monoclonal antibodies can bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies).Isolated and subcloned hybridoma cells or isolated phage, for example,can serve as a source of such DNA. Once isolated, the DNA can be placedinto expression vectors, which are then transfected into prokaryotic oreukaryotic host cells such as E. coli cells, simian COS cells, ChineseHamster Ovary (CHO) cells or myeloma cells that do not otherwise produceimmunoglobulins. More particularly, the isolated DNA (which can besynthetic as described herein) can be used to clone constant andvariable region sequences for the manufacture antibodies as described inNewman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which isincorporated by reference herein. Transformed cells expressing thedesired antibody can be grown up in relatively large quantities toprovide clinical and commercial supplies of the immunoglobulin.

In one embodiment, an isolated antibody or antigen-binding fragmentthereof comprises at least one heavy or light chain CDR of an antibodymolecule, e.g., A9E8. In another embodiment, an isolated antibody orantigen-binding fragment thereof comprises at least two CDRs from one ormore antibody molecules, e.g., A9E8. In another embodiment, an isolatedantibody or antigen-binding fragment thereof comprises at least threeCDRs from one or more antibody molecules, e.g., A9E8. In anotherembodiment, an isolated antibody or antigen-binding fragment thereofcomprises at least four CDRs from one or more antibody molecules, e.g.,A9E8. In another embodiment, an isolated antibody or antigen-bindingfragment thereof comprises at least five CDRs from one or more antibodymolecules, e.g., A9E8. In another embodiment, an isolated antibody orantigen-binding fragment thereof of the description comprises at leastsix CDRs from one or more antibody molecules, e.g., A9E8.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains can be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell-known in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs can be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody. Theframework regions can be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., J. Mol. Biol. 278:457-479 (1998) for a listing of human frameworkregions). The polynucleotide generated by the combination of theframework regions and CDRs encodes an antibody that specifically bindsto at least one epitope of a desired antigen, e.g., Siglec-15. One ormore amino acid substitutions can be made within the framework regions,and, the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods can be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentdisclosure and are within the capabilities of a person of skill of theart.

Also provided are antibodies or antigen-binding fragments thereof thatcomprise, consist essentially of, or consist of, variants (includingderivatives) of antibody molecules (e.g., the VH regions and/or VLregions) described herein, which antibodies or antigen-binding fragmentsspecifically bind to Siglec-15. Standard techniques known to those ofskill in the art can be used to introduce mutations in the nucleotidesequence encoding an antibody or antigen-binding fragment whichspecifically binds to Siglec-15, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. The variants (including derivatives) encodepolypeptides comprising less than 50 amino acid substitutions, less than40 amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference VH region, VHCDR1,VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a side chain with a similarcharge. Families of amino acid residues having side chains with similarcharges have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for biological activity toidentify mutants that retain activity (e.g., the ability to bindSiglec-15).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations can alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein can routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to bind at least one epitope ofSiglec-15) can be determined using techniques described herein or byroutinely modifying techniques known in the art.

III. Antibody Polypeptides and Immunoconjugates

The disclosure is further directed to isolated polypeptides which makeup antibodies or antigen-binding fragments thereof, which specificallybind to Siglec-15 and polynucleotides encoding such polypeptides.Isolated antibodies or fragments thereof as disclosed herein, comprisepolypeptides, e.g., amino acid sequences encoding, for example,Siglec-15-specific antigen binding regions derived from immunoglobulinmolecules. A polypeptide or amino acid sequence “derived from” adesignated protein refers to the origin of the polypeptide. In certaincases, the polypeptide or amino acid sequence which is derived from aparticular starting polypeptide or amino acid sequence has an amino acidsequence that is essentially identical to that of the starting sequence,or a portion thereof, wherein the portion consists of at least 10-20amino acids, at least 20-30 amino acids, at least 30-50 amino acids, orwhich is otherwise identifiable to one of ordinary skill in the art ashaving its origin in the starting sequence.

Also disclosed is an isolated antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15 comprising animmunoglobulin heavy chain variable region (VH) amino acid sequence atleast 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 1 as shown inTable 1.

Further disclosed is an isolated antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15 comprising a VH amino acidsequence identical to, or identical except for one, two, three, four,five, or more amino acid substitutions to SEQ ID NO: 1 as shown in Table1.

Also disclosed is an isolated antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15 comprising animmunoglobulin light chain variable region (VL) amino acid sequence atleast 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 2 as shown inTable 1.

Some embodiments disclose an isolated antibody or antigen-bindingfragment thereof which specifically binds to Siglec-15 comprising a VLamino acid sequence identical to, or identical except for one, two,three, four, five, or more amino acid substitutions to SEQ ID NO: 2 asshown in Table 1.

Also disclosed is an isolated antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15 comprising a VH-CDR1,VH-CDR2 and/or VH-CDR3, where the VH-CDR1, VH-CDR2 and VH-CDR3correspond to SEQ ID NO: 3, SEQ ID NO:4 and SEQ ID NO: 5, respectively.In further embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VL-CDR1, VL-CDR2 and/or VL-CDR3, where theVL-CDR1, VL-CDR2 and VL-CDR3 correspond to SEQ ID NO: 6, SEQ ID NO:7 andSEQ ID NO: 8, respectively. In particular embodiments, the isolatedantibody or antigen-binding fragment thereof comprises a VH-CDR1,VH-CDR2 VH-CDR3, VL-CDR1, VL-CDR2 and/or VL-CDR3, where the VH-CDR1,VH-CDR2 VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 correspond to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5. SEQ ID NO: 6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

Also disclosed is an isolated antibody or antigen-binding fragmentthereof which specifically binds to Siglec-15 comprising a VH-CDR1,VH-CDR2 and/or VH-CDR3; a VL-CDR1, VL-CDR2 and/or VL-CDR3; or a VH-CDR1,VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and/or VL-CDR3; where the sequence ofeach CDR is identical to, or identical to except for one, two or threeamino acid substitutions in each CDR relative to the VH-CDR1, VH-CDR2,VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 sequences corresponding to SEQ IDNO: 3, SEQ ID NO:4, SEQ ID NO: 5. SEQ ID NO: 6, SEQ ID NO:7 and SEQ IDNO: 8, respectively.

In certain embodiments, an isolated antibody or antigen-binding fragmentthereof as described herein specifically binds to Siglec-15 and causesan increased rate of endocytosis of the receptor. In some embodiments,the half-life of Siglec-15 endocytosis is less than about 5 minutes,about 4 minutes, about 3 minutes, about 2 minutes, about 1 minutes orabout 30 seconds in the human myelogenous leukemia cell line K562.

TABLE 1 Reference VH and VL amino acid sequences Antibody Name VHVH-CDR1 VH-CDR2 VH-CDR3 A9E8 QLQLQESGPGLVKPSET NWWS EVHHSGVT EFADDAFDLSLTCAVSGASISNWW (SEQ ID TYKPSLKS I SWVRQPPGKGLEWIGE NO: 3) (SEQ ID(SEQ ID VHHSGVTTYKPSLKSR NO: 4) NO: 5) VTISVDNSKNQLSLKLTSVTAADTAVYYCAREF ADDAFDIWGRGTMVTV SS (SEQ ID NO: 1) VL VL-CDR1 VL-CDR2VL-CDR3 SSELTQDPAVSVALGQT RGDSLRKY HKNNRAS NSRDTSGN VRITCRGDSLRKYYAS YAS(SEQ ID YLV WYQQKPRQAPQLVIYH (SEQ ID NO: 7) (SEQ ID KNNRASGIPDRFSGSISNO: 6) NO: 8) GNTASLTITGAQAEDEA AYFCNSRDTSGNYLVF GGGTKVTVLG(SEQ ID NO: 2)

Any anti-Siglec-15 antibodies or fragments, variants or derivativesthereof described herein can further include additional polypeptides,e.g., a signal peptide to direct secretion of the encoded polypeptide,antibody constant regions as described herein, or other heterologouspolypeptides as described herein. Additionally, antibodies or fragmentsthereof of the description include polypeptide fragments as describedelsewhere. Additionally anti-Siglec-15 antibodies or fragments, variantsor derivatives thereof described herein can be fusion polypeptides, Fabfragments, scFvs, or other derivatives, as described herein.

Also, as described in more detail elsewhere herein, the disclosureincludes compositions comprising anti-Siglec-15 antibodies or fragments,variants or derivatives thereof described herein.

It will also be understood by one of ordinary skill in the art thatanti-Siglec-15 antibodies or fragments, variants or derivatives thereofdescribed herein can be modified such that they vary in amino acidsequence from the naturally occurring binding polypeptide from whichthey were derived. For example, a polypeptide or amino acid sequencederived from a designated protein can be similar, e.g., have a certainpercent identity to the starting sequence, e.g., it can be 60%, 70%,75%, 80%, 85%, 90%, or 95% identical to the starting sequence.

As known in the art, “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, whether anyparticular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95%identical to another polypeptide can be determined using methods andcomputer programs/software known in the art such as, but not limited to,the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981),to find the best segment of homology between two sequences. When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for example, 95% identical to a referencesequence, the parameters are set, of course, such that the percentage ofidentity is calculated over the full length of the reference polypeptidesequence and that gaps in homology of up to 5% of the total number ofamino acids in the reference sequence are allowed.

Furthermore, nucleotide or amino acid substitutions, deletions, orinsertions leading to conservative substitutions or changes at“non-essential” amino acid regions can be made. For example, apolypeptide or amino acid sequence derived from a designated protein canbe identical to the starting sequence except for one or more individualamino acid substitutions, insertions, or deletions, e.g., one, two,three, four, five, six, seven, eight, nine, ten, fifteen, twenty or moreindividual amino acid substitutions, insertions, or deletions. Incertain embodiments, a polypeptide or amino acid sequence derived from adesignated protein has one to five, one to ten, one to fifteen, or oneto twenty individual amino acid substitutions, insertions, or deletionsrelative to the starting sequence.

An anti-Siglec-15 antibody or fragment, variant or derivative thereofdescribed herein can comprise, consist essentially of, or consist of afusion protein. Fusion proteins are chimeric molecules which comprise,for example, an immunoglobulin antigen-binding domain with at least onetarget binding site, and at least one heterologous portion, i.e., aportion with which it is not naturally linked in nature. The amino acidsequences can normally exist in separate proteins that are broughttogether in the fusion polypeptide or they can normally exist in thesame protein but are placed in a new arrangement in the fusionpolypeptide. Fusion proteins can be created, for example, by chemicalsynthesis, or by creating and translating a polynucleotide in which thepeptide regions are encoded in the desired relationship.

The term “heterologous” as applied to a polynucleotide, polypeptide, orother moiety means that the polynucleotide, polypeptide, or other moietyis derived from a distinct entity from that of the rest of the entity towhich it is being compared. In a non-limiting example, a “heterologouspolypeptide” to be fused to an antibody or an antigen-binding fragment,variant, or derivative thereof is derived from a non-immunoglobulinpolypeptide of the same species, or an immunoglobulin ornon-immunoglobulin polypeptide of a different species.

Toxins can be employed with antibodies or polypeptides of the presentinvention to yield immunoconjugates. Exemplary toxins include ricin,abrin, diptheria toxin and subunits thereof, as well as botulinum toxinsA through F. These toxins are readily available from commercial sources(e.g., Sigma Chemical Company, St. Louis, Mo.). Diptheria toxin isisolated from Corynebacterium diphtheriae. Ricin is the lectin RCA60from Ricinus communes (Castor bean). The term also references toxicvariants thereof. See, U.S. Pat. Nos. 5,079,163 and 4,689,401. Ricinuscommunis agglutinin (RCA) occurs in two forms designated RCA₆₀ andRCA₁₂₀ according to their molecular weights of approximately 65,000 and120,000, respectively. Nicholson and Blaustein, J. Biochim. Biophys.Acta, 266:543 (1972). The A chain is responsible for inactivatingprotein synthesis and killing cells. The B chain binds ricin tocell-surface galactose residues and facilitates transport of the A chaininto the cytosol (Olsnes et al., Nature, 1974; 249:627-631). See, U.S.Pat. No. 3,060,165.

In one embodiment, the toxin is Pseudomonas exotoxin. Pseudomonasexotoxin A (PE) is an extremely active monomeric protein (molecularweight 66 kD), secreted by Pseudomonas aeruginosa, which inhibitsprotein synthesis in eukaryotic cells through the inactivation ofelongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The Pseudomonas exotoxins (PE) can include the native sequence,cytotoxic fragments of the native sequence, and conservatively modifiedvariants of native PE and its cytotoxic fragments. Cytotoxic fragmentsof PE include those which are cytotoxic with or without subsequentproteolytic or other processing in the target cell (e.g., as a proteinor pre-protein). Cytotoxic fragments of PE include PE40, PE38, and PE35.PE40 is a truncated derivative of PE as previously described in the art.See, Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-62 (1991); Kondo etal., J. Biol. Chem. 263:9470-9475 (1988). PE38 is a truncated PEcomposed of amino acids 253-364 and 381-613. PE35 is a 35 kDcarboxyl-terminal fragment of PE composed of a Met at position 280followed by amino acids 281-364 and 381-613 of native PE. In preferredembodiments, the cytotoxic fragment PE38 is employed. PE38 is apro-protein which can be activated to its cytotoxic form upon processingwithin a cell.

With the Pseudomonas exotoxins and antibodies herein provided, one ofskill can readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same PE or antibody sequence. Thus, the presentinvention provides nucleic acids encoding antibodies and conjugates andfusions thereof.

In other embodiments the toxin is a holotoxin (e.g., a ribosomeinactivating protein type II) or a hemitoxin (e.g., saporin, gelonin).In further embodiments, the toxin is a cytotoxic agent or compound,examples of which include taxanes, DNA-alkylating agents,anthracyclines, microtubule inhibitors (e.g., tubulysins), duocarmycins,maytansinoids, doxorubicin and auristatins.

IV. Polynucleotides Encoding Antibodies or Antigen-Binding FragmentsThereof

Also provided herein are nucleic acid molecules encoding theanti-Siglec-15 antibodies or fragments, variants or derivatives thereofdescribed herein.

One embodiment provides an isolated polynucleotide comprising,consisting essentially of, or consisting of a nucleic acid encoding animmunoglobulin heavy chain variable region (VH) amino acid sequence atleast 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 1 as shown inTable 1.

Another embodiment provides an isolated polynucleotide comprising,consisting essentially of, or consisting of a nucleic acid encoding a VHamino acid sequence identical to, or identical except for one, two,three, four, five, or more amino acid substitutions to SEQ ID NO: 1 asshown in Table 1.

A further embodiment provides an isolated antibody or antigen-bindingfragment comprising the VH encoded by the polynucleotide specifically orpreferentially binds to Siglec-15.

Another embodiment provides an isolated polynucleotide comprising,consisting essentially of, or consisting of a nucleic acid encoding animmunoglobulin light chain variable region (VL) amino acid sequence atleast 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 2 as shown inTable 1.

A further embodiment provides an isolated polynucleotide comprising,consisting essentially of, or consisting of a nucleic acid encoding a VLamino acid sequence identical to, or identical except for one, two,three, four, five, or more amino acid substitutions to SEQ ID NO: 2 asshown in Table 1.

In another embodiment, an isolated antibody or antigen-binding fragmentcomprising the VL encoded by the polynucleotide specifically orpreferentially binds to Siglec-15.

One embodiment provides an isolated polynucleotide comprising,consisting essentially of, or consisting of a nucleic acid at least 80%,85%, 90% 95% or 100% identical to SEQ ID NO:3 or SEQ ID NO:4 as shown inTable 2.

TABLE 2 Reference nucleic acid sequences Antibody Namenucleotide sequences A9E8 - VHCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAAACCCTGTCCCTCACCTGCGCTGTCTCTGGTGCCTCCATTAGTAACTGGTGGACTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAGTCCATCATAGTGGAGTCACCACCTACAAGCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAACTCGAAGAACCAATTATCTCTGAAGCTAACCTCTGTGACAGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGAGTTCGCGGATGATGCTTTTGATATCTGGGGCCGAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 3) A9E8 - VLTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTGGGACAGACAGTCAGGATCACATGCCGAGGGGACAGCCTCAGAAAGTATTATGCAAGCTGGTACCAGCAGAAGCCACGACAGGCCCCTCAACTTGTCATCTATCATAAAAACAACAGGGCGTCAGGGATCCCAGACCGATTCTCTGGCTCCATCTCCGGAAACACAGCTTCTTTGACCATCACTGGGGCTCAGGCAGAAGATGAGGCTGCCTATTTCTGTAATTCTCGGGACACCAGTGGTAATTATCTGGTCTTCGGCGGAGGGACCAAGGTC ACCGTCCTAGGT(SEQ ID NO: 4)

In some embodiments, an isolated antibody or antigen-binding fragmentthereof encoded by one or more of the polynucleotides described above,which specifically binds to Siglec-15, comprises, consists essentiallyof, or consists of VH and VL amino acid sequences at least 80%, 85%, 90%95% or 100% identical to SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The disclosure also includes fragments of the polynucleotides asdescribed elsewhere herein. Additionally polynucleotides which encodefusion polynucleotides, Fab fragments, and other derivatives, asdescribed herein, are also provided.

The polynucleotides can be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody can be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al., BioTechniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an anti-Siglec-15 antibody orfragment, variant or derivative thereof can be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody can bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably poly A+RNA, isolated from, any tissue or cellsexpressing the antibody or such as hybridoma cells selected to expressan antibody) by PCR amplification using synthetic primers hybridizableto the 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR can then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of ananti-Siglec-15 antibody or fragment, variant or derivative thereof isdetermined, its nucleotide sequence can be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

A polynucleotide encoding an anti-Siglec-15 antibody or fragment,variant or derivative thereof can be composed of any polyribonucleotideor polydeoxyribonucleotide, which can be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding ananti-Siglec-15 antibody or fragment, variant or derivative thereof canbe composed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an anti-Siglec-15antibody or fragment, variant or derivative thereof can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an anti-Siglec-15 antibody or fragment, variantor derivative thereof can also contain one or more modified bases or DNAor RNA backbones modified for stability or for other reasons. “Modified”bases include, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions are made at one or morenon-essential amino acid residues.

V. Expression of Antibody Polypeptides

As is well known, RNA can be isolated from the original hybridoma cellsor from other transformed cells by standard techniques, such asguanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA can be isolatedfrom total RNA by standard techniques such as chromatography on oligo dTcellulose. Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains ofthe anti-Siglec-15 antibody or fragment, variant or derivative thereofcan be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well-known methods.PCR can be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also can be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries can be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, can be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA can be synthetic according to the presentdisclosure at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provide ananti-Siglec-15 antibody or fragment, variant or derivative thereof ofthe disclosure, the polynucleotides encoding anti-Siglec-15 antibodiesor antigen-binding fragments thereof are typically inserted in anexpression vector for introduction into host cells that can be used toproduce the desired quantity of anti-Siglec-15 antibodies orantigen-binding fragments thereof.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule described herein, e.g., Siglec-15, requires constructionof an expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (containing the heavyor light chain variable domain), of the disclosure has been obtained,the vector for the production of the antibody molecule can be producedby recombinant DNA technology using techniques well known in the art.Thus, methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Thedisclosure, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the disclosure, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors can include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody can be cloned intosuch a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present disclosure as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors can easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant disclosure will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this disclosure, numerous expression vector systemscan be employed. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomescan be selected by introducing one or more markers which allow selectionof transfected host cells. The marker can provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements can also beneeded for optimal synthesis of mRNA. These elements can include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In some embodiments the cloned variable region genes are inserted intoan expression vector along with the heavy and light chain constantregion genes (e.g., human) synthetic as discussed above. Of course, anyexpression vector which is capable of eliciting expression in eukaryoticcells can be used in the present disclosure. Examples of suitablevectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo,pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV,pUB6/V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego,Calif.), and plasmid pCI (available from Promega, Madison, Wis.). Ingeneral, screening large numbers of transformed cells for those whichexpress suitably high levels if immunoglobulin heavy and light chains isroutine experimentation which can be carried out, for example, byrobotic systems.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the anti-Siglec-15 antibody or fragment, variant orderivative thereof of the disclosure has been prepared, the expressionvector can be introduced into an appropriate host cell. Introduction ofthe plasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors”Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass.,Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction intothe host is via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the disclosure includes host cells containing apolynucleotide encoding anti-Siglec-15 antibody or fragment, variant orderivative thereof, or a heavy or light chain thereof, operably linkedto a heterologous promoter. In some embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains can be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

Certain embodiments include an isolated polynucleotide comprising anucleic acid which encodes the above-described VH and VL, wherein anantibody or antigen-binding fragment thereof expressed by thepolynucleotide specifically binds Siglec-15. In some embodiments thepolynucleotide as described encodes an scFv molecule including VH andVL, at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO:3 or SEQID NO:4 as shown in Table 2.

Some embodiments include vectors comprising the above-describedpolynucleotides. In further embodiments, the polynucleotides areoperably associated with a promoter. In additional embodiments, thedisclosure provides host cells comprising such vectors. In furtherembodiments, the disclosure provides vectors where the polynucleotide isoperably associated with a promoter, wherein vectors can express anantibody or antigen-binding fragment thereof which specifically bindsSiglec-15 in a suitable host cell.

Also provided is a method of producing an antibody or fragment thereofwhich specifically binds Siglec-15, comprising culturing a host cellcontaining a vector comprising the above-described polynucleotides, andrecovering said antibody, or fragment thereof. In further embodiments,the disclosure provides an isolated antibodies or fragments thereofproduced by the above-described method.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” can mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems can be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe disclosure in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant east expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichiacoli, or eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled n the art are credited with ability to determineparticular host cell lines which are best suited for the desired geneproduct to be expressed therein. Exemplary host cell lines include, butare not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB 11(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervicalcarcinoma), CV1 (monkey kidney line), COS (a derivative of CV1 with SV40T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610(Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamsterkidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma),BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and 293(human kidney). Host cell lines re typically available from commercialservices, the American Tissue Culture Collection or from publishedliterature.

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule can be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Constructs encoding anti-Siglec-15 antibodies or fragments, variants orderivatives thereof, as disclosed herein can also be expressednon-mammalian cells such as bacteria or yeast or plant cells. Bacteriawhich readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologouspolypeptides must be isolated,purified and then assembled into functional molecules. Where tetravalentforms of antibodies are desired, the subunits will then self-assembleinto tetravalent antibodies (WO02/096948A2).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence can be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes can also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once the anti-Siglec-15 antibody or fragment, variant or derivativethereof, as disclosed herein has been recombinantly expressed, it can bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

VI. Pharmaceutical Compositions Comprising Anti-Siglec-15 Antibodies orAntigen-Binding Fragments Thereof

The pharmaceutical compositions used in this disclosure comprisepharmaceutically acceptable carriers well known to those of ordinaryskill in the art. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Certain pharmaceutical compositions as disclosed herein can be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also can be administered by nasal aerosol or inhalation.Preservatives and other additives can also be present such as forexample, antimicrobials, antioxidants, chelating agents, and inert gasesand the like. Suitable formulations for use in the therapeutic methodsdisclosed herein are described in Remington's Pharmaceutical Sciences,Mack Publishing Co., 16th ed. (1980).

The amount of an anti-Siglec-15 antibody or immunoconjugate, that can becombined with the carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. Dosage regimens also can be adjusted to provide theoptimum desired response. The compositions can also comprise theSiglec-15 immunoconjugates, antibodies or fragments, variants orderivatives thereof dispersed in a biocompatible carrier material thatfunctions as a suitable delivery or support system for the compounds.

VII. Treatment Methods Using Therapeutic Immunoconjugates, Antibodies orAntigen-Binding Fragments Thereof

Methods of preparing and administering a Siglec-15 an immunoconjugate,antibody or fragment, variant or derivative thereof, as disclosed hereinto a subject in need thereof are well known to or are readily determinedby those skilled in the art.

Siglec-15 immunoconjugates, antibodies or antigen-binding fragmentsthereof of the invention are useful for the treatment of a leukemia,such as AML. In certain embodiments, the Siglec-15 immunoconjugates,antibodies or antigen-binding fragments thereof advantageously reduceside effects associated with conventional leukemic therapies.

Leukemia's are cancers that originate in the bone marrow, where themalignant cells are white blood cells (leukocytes). Acute myelogenousleukemia (also called acute myelocytic leukemia, acute myeloblasticleukemia, acute granulocytic leukemia, and acute nonlymphocyticleukemia) is a malignancy that arises in either granulocytes ormonocytes. AML is characterized by the uncontrolled, exaggerated growthand accumulation of cells called leukemic blasts, which fail to functionas normal blood cells, and the blockade of the production of normalmarrow cells, leading to a deficiency of red cells (anemia), andplatelets (thrombocytopenia) and normal white cells (especiallyneutrophils, i.e., neutropenia) in the blood.

CD33-targeted therapies, such as GO, produce significant side effectsincluding an infusion syndrome when administered to AML patients. Thus,the ability to target Siglec-15 for therapy provides an alternative toCD33-based treatments. Moreover, since leukemic blasts are both CD33+and CD33−, anti-Siglec-15 antibodies or antigen-binding fragmentsthereof provide an additional treatment option for AML. This isimportant for several reasons. Improved cell killing can increase thepercentage of complete remissions, increase remission time, and decreasethe likelihood of relapse. Secondly, improved cell killing can decreasethe total requirement for toxin therapy, resulting in decreasednonspecific cell killing and side effects. Thirdly, for those AMLpatients who have relatively low expression of CD33, either in thenatural state or due to outgrowth of CD33− cells after undergoing CD33therapy, Siglec-15 antibodies or antigen-binding fragments thereof arevaluable alternative or combination therapeutics.

Moreover, since surface expression of Siglec-15 is low on peripheralblood leukocytes in normal control donors and high on blasts from AMLpatient, the side effects from administration of Siglec-15immunoconjugates, antibodies or antigen-binding fragments thereof willbe reduced compared to conventional leukemic therapies.

All subtypes of AML are suitable for treatment with an Siglec-15antibody or antigen-binding fragment thereof. The subtypes of AML areclassified based on the stage of development myeloblasts have reached atthe time of diagnosis. The categories and subsets allow the physician todecide what treatment works best for the cell type and how quickly thedisease may develop. The subsets are: M0, myeloblastic, on specialanalysis; M1, Myeloblastic, without maturation; M2, Myeloblastic, withmaturation; M3, Promyelocytic; M4, Myelomonocytic; M5, Monocytic; M6,Erythroleukemia; and M7, Megakaryocytic. A Siglec-15 antibody orantigen-binding fragment thereof can be administered with a secondaryagent that is particularly suited to the subtype of AML. For example,acute promyelocytic leukemia (APL) and acute monocytic leukemia aresubtypes of AML that need different treatment than other subtypes ofAML. A second agent for treatment of APL can include all-trans retinoicacid (ATRA) or an antimetabolite, such as cytarabine. A second agent fortreatment of acute monocytic leukemia can include a deoxyadenosineanalog, such as 2-chloro-2′-deoxyadenosine (2-CDA).

Risk factors of AML include the presence of certain genetic disorders,such as Down syndrome, Fanconi anemia, Shwachman-Diamond syndrome andothers. A patient having AML and a genetic disorder can be administereda Siglec-15 antibody or antigen-binding fragment thereof and a secondagent to treat a symptom of the genetic disorder. For example, a patientwith AML and Fanconi anemia can be administered a Siglec-15 antibody orantigen-binding fragment thereof and an antibiotic.

Other risk factors for AML include chemotherapy or radiotherapy fortreatment of a different cancer, tobacco smoke, and exposure to largeamounts of benzene.

Siglec-15 immunoconjugates, antibodies or antigen-binding fragmentsthereof can be administered to a human or other animal in accordancewith the aforementioned methods of treatment in an amount sufficient toproduce a therapeutic effect. With respect to AML, therapy can be deemedto be effective if there is a statistically significant difference inthe rate or proportion of malignant cells in the blood stream or bonemarrow. Therapy is deemed to be effective, for example, when remissionis achieved, which is when there are no signs of malignant cells.

Efficacy of administering a first agent and, optionally, a second agent,can also be evaluated based on, for example, the decrease of number ofmalignant cells found in the blood stream, a decrease in frequency orseverity of bacterial or viral infection, increased rate of woundhealing, and the general feeling of the patient, including increasedenergy level and decreased soreness in bones and joints.

The route of administration of the anti-Siglec-15 immunoconjugate,antibody or fragment, variant or derivative thereof, can be, forexample, oral, parenteral, by inhalation or topical. The term parenteralas used herein includes, e.g., intravenous, intraarterial,intraperitoneal, intramuscular, or subcutaneous administration. Asuitable form for administration would be a solution for injection, inparticular for intravenous or intraarterial injection or drip.

Anti-Siglec-15 immunoconjugates, antibodies or antigen-binding fragmentsthereof can be administered multiple occasions at various frequenciesdepending on various factors known to those of skill in the art.Alternatively, anti-Siglec-15 immunoconjugates, antibodies orantigen-binding fragments thereof can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theimmunoconjugate, antibody, or antigen-binding fragment in the patient.

The practice of the disclosure will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring HarborLaboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6^(th) ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and ane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

EXAMPLES Example 1 Multiple Splicing and Transcript Induction ofSiglec-15 in Leukemic Cell Lines

Four human Siglec-15 splice variants were identified by RT-PCR usingspecific primers. Electrophoresis of RT-PCR products from DAMI(megakaryocytic cell line), HEL (erythroleukaemic cell line) and K562(erythroleukaemic cell line) are shown in FIG. 1A with exon mapsgenerated after sequencing (FIG. 1A—right panel). Four different splicevariants of Siglec-15 were identified from RT-PCR of cDNA from leukemiccell lines. The largest transcript (2D) comprised all exons and was usedin further experiments.

Siglec-15 transcript was also found in 6 of 7 different cell linestested (FIG. 1B). Only YT (natural killer-like leukemic cell line)showed no Siglec-15 transcript. Phorbomeristyl acetate (PMA) orlipopolysaccharide (LPS) stimulations over 48 hours up-regulatedexpression of the transcripts. GAPDH RT-PCR is shown underneath eachpanel to control for equal loading (0.4 kb). 293T cells were used as anegative control.

RT-PCR was also performed to identify Siglec-15 full length transcriptin 7 cell lines derived from leukemic patients (FIG. 1B). These celllines were also stimulated with either LPS or PMA for 48 hours.Siglec-15 transcript expression was found in 6 of 7 cell lines andup-regulation following stimulation was observed in all cases apart fromin the CHRF-288-11 cell line. The full length transcript was the onlyone to be upregulated consistently. In the HL60 cell line, while PMAinduced upregulation of the full-length transcript, LPS stimulationresulted in complete loss of this transcript. These results show thatSiglec-15 exists in multiple splice variants and the full lengthtranscript is inducible by LPS and PMA in many leukemic cell lines.

Example 2 Siglec-15 Antibodies or Antigen-Binding Fragments Thereof

Phage display was employed to select reagents that specifically bind toSiglec-15. Single chain fragment variable (ScFv) binders to purifiedFlag-His tagged Siglec-15 were amplified using phage display technologyand further selected on the surface of CHO cells stably expressingHA-tagged Siglec-15 cloned in a pDisplay construct. A9E8 and A4C9 weretwo ScFv binders from these two rounds that were converted to murineIgG1 format. Both bound Siglec-15 by FACS (FIG. 2A) and confocalmicroscopy (FIG. 2B) and A4C9, also bound in Western blots (FIG. 2D).

These two antibodies are unlikely to cross-react with other Siglecsbecause the similarity between Siglec-15 and other Siglecs is very low(identity less than 35%). To confirm specificity of A9E8 and A4C9 toSiglec-15, the ScFv versions of the two antibodies were compared fortheir binding to CHO cells stably transfected with either HA-taggedSiglec-15, Siglec-11, Siglec-16 or untransfected CHO cells. These twoantibodies bound Siglec-15 strongly, while no binding was observed forSiglec-11 and -16 transfectants (FIG. 2C). U937 is a well characterizedcell line that endogenously expresses many Siglecs on its cell surface,including CD33, Siglec-5 and Siglec-9 (Nguyen D H et al 2006). A9E8 didnot bind to U937 cell surface (FIG. 5A) again consistent with nocross-reactivity with other Siglecs.

Example 3 Siglec-15 Expression Patterns

It has been previously shown by immunohistochemistry that endogenousSiglec-15 is mostly intracellular (Angata et al 2007). This finding wasconfirmed by FACS analysis of unfixed as well as fixed and permeabilizedleukemic cell lines, although these lines differed in relative levels ofsurface and intracellular expression (FIG. 3). Low surface expressionmay be explained by the requirement of a membrane adaptor protein, suchas DAP12 and DAP10 (Angata et al 2007). To test for the requirement of amembrane adaptor protein, Flag-tagged Siglec-15 was transientlytransfected into HEK-293 cell lines stably transfected with differentadaptor molecules: DAP12, DAP10 or FcRγ. HEK-293 cells were chosenbecause they lack endogenous expression of these adaptors. SurfaceFlag-Siglec-15 expression was detected using anti-Flag monoclonalantibody by FACS. Increase in surface Siglec-15 was most notable inpresence of DAP10, but significant expression was also observed in thepresence of DAP12 and FcRγ (FIG. 4). A parallel experiment usingSiglec-16 showed more specific DAP12-dependent surface expression (FIG.4). These results show that Siglec-15 is a prominently intracellularantigen under normal growth conditions but some may access the cellsurface, in conjunction with an adaptor. The apparent promiscuity ofSiglec-15's dependence of adaptors for surface expression sets it apartfrom other activating receptors, which bind to one adaptor morespecifically.

The antibody A9E8 was used to examine Siglec-15 surface expression onleukemic cell lines. By FACS, Siglec-15 was identified on the cellsurface of leukemic cell lines of the erythroid (K562 and HEL) andmegakaryocytic (DAMI) lineages (FIG. 5A). Monocytic (U937 and SKM-1) andpromonocytic (NOMO-1) leukemic cell lines showed no cell surfaceexpression (FIG. 5A). Leukemic cell lines of lymphoid lineages were alsotested. Only Daudi (B cell lymphoma) showed, relatively weak, surfaceexpression (FIG. 5A). There was no expression on Jurkat T celllymphoblastic leukemia (FIG. 5A).

Western blot analysis of selected cell lines by monoclonal antibody A4C9showed a similar pattern of expression to a rabbit anti-serum, 1A,specific to the cytoplasmic tail of Siglec-15, which bound transfectantFlag-tagged Siglec-15 in 293T cells (FIGS. 5B and 5C). The western blotsrevealed significant Siglec-15 protein expression in Jurkat cells (bandindicated by single arrows) and weaker expression in K562 and HEL celllines (FIG. 5C).

Example 4 Siglec-15 Surface Expression on Peripheral Blood Leukocytesfrom Healthy Donors

Healthy donor peripheral blood cells were used for studying Siglec-15expression.

Siglec-15 was absent on the surface of most mature leukocytes tested: Tcells (CD3+), B cells (CD19+) and NK cells (CD56+) but rare populations(<1%) of monocytes (CD14+) showed Siglec-15 surface expression (FIG.6A). This pattern of expression was similar in six healthy donorstested.

Siglec-15 was previously reported to be intracellular in macrophages anddendritic cells by immunohistochemistry. FACS analysis showed very weaklevels of Siglec-15 surface expression on 7% of cultured macrophagesderived from peripheral blood monocytes (FIG. 6B). The Siglec-15staining was more prominent on cells of low forward scatter, probablycorresponding to a less mature subpopulation of macrophages. The surfacestaining on macrophages was lost following 24 hours of LPS treatment(FIG. 6B). Dendritic cells showed extremely low Siglec-15 surfaceexpression on ˜4% of the total population before LPS stimulation (FIG.6B). This expression was also lost following 24 hours of LPS stimulation(FIG. 6B). These results show that in healthy donors, Siglec-15 is lowor absent on the cell surface of most blood circulating leukocytes, asit was on macrophages and dendritic cells derived from monocytes.

Example 5 Siglec-15 Surface Expression on Circulating AML Blasts

In contrast to healthy donors, significantly higher Siglec-15 surfaceexpression was found on blasts in 8 out of 10 AML patients tested (FIG.7A-D). The average size of blast subpopulations positive for Siglec-15was 20%, compared to ˜2% for circulating peripheral blood leukocytesfrom healthy donors (FIG. 7E). Four examples representative of the groupof AML donors studied are summarized in FIG. 7. Patients A (FIG. 7A), B(FIG. 7B) and C (FIG. 7C) consistently showed co-expression of Siglec-15with CD14.

CD33 is expressed on 90% of AML myeloblasts. In one case, patient C(FIG. 7C), who was CD33-negative, a high level of Siglec-15 wasexpressed on 20% of blasts. In another case, patient D showed a lowpercentage of CD33+ blasts (7%) but a higher proportion of Siglec-15+blasts (15%) (data not shown). HLA-DR was not consistently co-expressedwith Siglec-15, except in certain patients, such as A. Patient Dexpressed more Siglec-15 on larger blasts, as defined by greater forwardscatter. These data indicate that Siglec-15 exhibits significantlyhigher expression on circulating white cells from AML patients than fromhealthy donors.

Example 6 A9E8 Induces an Extremely Rapid Rate of Endocytosis of SurfaceSiglec-15

A fast rate of Siglec-15 endocytosis would be advantageous particularlyif a toxin-conjugated antibody were to be used to target Siglec-15positive AML blasts. Rapid endocytosis of Siglec-15 is expected from itscytoplasmic tail sequence, which encodes an ITSM motif of sequenceSNYENL and therefore conforms to the classical endocytosis YxxΦ motif,where X is any amino acid and Φ is a hydrophobic residue. The YxxΦ motifdirects clathrin-dependent endocytosis of membrane proteins (Collawn etal 1990) by binding to the AP2 clathrin adaptor complex (Aguilar et al2001; Boehm and Bonifacino, 2001). To investigate the rate of endogenousSiglec-15 endocytosis in vitro, the K562 cell line was chosen because itshows significant surface expression. The A9E8 antibody was bound toK562 on ice and cells were resuspended in media pre-warmed to 37° C.Warmed cells were immediately incubated for various times at 37° C.,before cooling with 5 ml of ice-cold FACS buffer. Secondary antibody wasused to detect remaining surface antibody and the half-life ofendocytosis was measured at 174 seconds. To control for naturaldissociation of the A9E8 antibody from Siglec-15, a pDisplay constructencoding only the extracellular domains of Siglec-15 was subjected tothe same assay. Lacking the natural transmembrane and cytoplasmic tailof Siglec-15, this construct exhibited no apparent surface endocytosisduring the first fifteen minutes of incubation at 37° C. This resultshows that disappearance of surface A9E8 staining on K562 cells is mostlikely due to endocytosis.

The disclosure is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the disclosure, and any compositions or methods which arefunctionally equivalent are within the scope of this disclosure. Indeed,various modifications of the disclosure in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. An isolated antibody or antigen-binding fragment thereof thatspecifically binds to Siglec-15 comprising: (a) a VH sequence at least90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO:1;(b) a VL sequence at least 90%, 95% or 100% identical to the amino acidsequence of SEQ ID NO:2; (c) a VH-CDR1, VH-CDR2 and/or VH CDR3 sequenceidentical to or identical to except for one, two, or three substitutionsin each CDR relative to the VH-CDR1, VH-CDR2 and VH-CDR3 sequencescorresponding to SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively;(d) a VL-CDR1, VL-CDR2 and/or VL CDR3 sequence identical to or identicalto except for one, two, or three substitutions in each CDR relative tothe VL-CDR1, VL-CDR2 and VL-CDR3 sequences corresponding to SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8, respectively; or (e) VH-CDR1, VH-CDR2, VHCDR3, VL-CDR1, VL-CDR2 and VL CDR3 sequences identical to or identicalto except for one, two, or three substitutions in each CDR relative tothe VH-CDR1, VH-CDR2, VH CDR3, VL-CDR1, VL-CDR2 and VL CDR3 sequencescorresponding to SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8, respectively.
 2. An isolated antibody orantigen-binding fragment thereof according to claim 1, wherein saidantibody or antigen-binding fragment thereof induces endocytosis ofSiglec-15 at a rate where the half-life of endogenous surface Siglec-15on human myelogenous leukemia K562 cells is less than about 5 minutes,less than about 4 minutes, or less than about 3 minutes. 3-7. (canceled)8. The antibody or fragment thereof of claim 2, wherein the VH and/or VLsequences are at least 95% identical to the reference VH of SEQ ID NO:1and at least 95% identical to the reference VL sequence of SEQ ID NO:2.9-10. (canceled)
 11. An isolated antibody or antigen-binding fragmentthereof which specifically binds to the same Siglec-15 epitope as anantibody or antigen-binding fragment according to claim
 1. 12.(canceled)
 13. An isolated antibody or antigen-binding fragment thereofwhich specifically binds to Siglec-15, and competitively inhibitsSiglec-15 binding by an antibody or antigen-binding fragment thereofcomprising the VH and/or VL of A9E8.
 14. The antibody or fragmentthereof of claim 1, which is humanized, chimeric, fully human, a Fabfragment, a Fab′ fragment, a F(ab)2 fragment, or a single chain Fv(scFv) fragment.
 15. A polypeptide comprising the VH and/or VL sequencesof claim
 1. 16. An isolated cell producing the antibody or fragmentthereof of claim
 1. 17. A method of making the antibody or fragmentthereof of claim 1, comprising (a) culturing an isolated cell producingthe antibody or fragment thereof; and (b) isolating said antibody orfragment thereof from said cultured cell.
 18. (canceled)
 19. Animmunoconjugate having the formula (A)-(L)-(C) or (C)-(L)-(A), wherein:(A) is an antibody or antigen binding fragment thereof, of claim 1; (L)is a linker; and (C) is a cytotoxic agent; and wherein said linker (L)links (A) to (C).
 20. A composition comprising the antibody or fragmentthereof of claim 1 and a carrier.
 21. An isolated polynucleotidecomprising a nucleic acid encoding the VH and/or VL of an antibody orfragment thereof of claim
 1. 22. The polynucleotide of claim 21 whichencodes an antibody comprising the nucleotide sequence SEQ ID NO: 9 orSEQ ID NO:
 10. 23. A vector comprising the polynucleotide of claim 21.24. A method of treating acute myeloid leukemia in a subject in needthereof, comprising administering to the subject an effective amount ofan antibody or fragment thereof, wherein said antibody or fragmentthereof specifically binds to Siglec-15.
 25. The method of claim 24comprising administering to the subject an effective amount of anantibody or fragment thereof of claim
 1. 26. (canceled)
 27. A method ofreducing the side effects associated with treating acute myeloidleukemia in a subject in need thereof, comprising administering to thesubject an effective amount of an antibody or fragment thereof ofclaim
 1. 28-29. (canceled)
 30. A method of reducing the number of blastsin an AML patient comprising administering to the subject an effectiveamount of an antibody or fragment thereof of claim
 1. 31-34. (canceled)35. A method of treating acute myeloid leukemia in a subject havingeither low expression of CD33 or are CD33−, comprising administering tothe subject an effective amount of an antibody or fragment thereof ofclaim
 1. 36-37. (canceled)
 38. A method for making acute myeloidleukemia therapeutics comprising generating antibodies that bindSiglec-15, wherein said therapeutics preferentially target AML blastsover normal peripheral blood leukocytes.
 39. (canceled)